1. Field of the Invention
The present invention relates to the treatment of allergies. Allergic disorders affect as many as one in four Americans (59,000,000). More than 17% have upper respiratory allergies, including hay fever. Another 4% have asthma, and 10% have allergic skin conditions such as eczema and other rashes. It is estimated that some 40,000,000 school days are lost yearly because of asthma, in addition, approximately 6,000 lives are lost to asthma, and dozens die of insect bites, annually. The accepted allergy treatment method is hyposensitization injections containing minute amounts of allergens causing the reaction, given in incremental doses once or twice weekly until symptoms improve, and then maintained at a set dosage with decreasing frequency for approximately three years. If carefully selected, approximately one-third of patients find their symptoms are relieved substantially by this therapy. The rest have mild to moderate improvement. This immunotherapy method has been standard practice since 1911. The present invention provides a method of immunotherapy and a modified allergen extract and a method of making the modified allergen extract.
2. Description of the Prior Act
Modified allergen extracts for use in immunotherapy have been reported in the literature. Such extracts have been modified with the desire to reduce their allergenicity without sacrificing immunogenicity and hence achieve improved immunotherapy results with fewer injections. Polymerized grass pollen allergens have been prepared and tested in immunotherapy. See, Patterson et al., "Polymerization of Individual Species of Grass Pollen Allergen", J. Allergy Clin. Immunol 72:129-133, (1983); Fitzsimons et al., "A comparison of the Immune Response to Immunotherapy with Polymerized Grass Allergen and Monomeric Grass Allergen"; Annals of Allergy 57:291-294 (1986); Fitzsimons et al., supra reports that polymerized and monomeric grass pollen extract give comparable result in immunotherapy. Further reports have suggested that the polymerized grasses contain an increased amount of immunity producing allergen. It has been reported that in use a series of fifteen (15) injections with the polymerized pollen are as effective as a series of seventy (70) standard injections. Modified allergens for allergy desensitization have also been prepared by treating pollen extract with formaldehyde. Such formaldehyde treated allergens are referred to as "allergoids". Polyethylene glycol modified ragweed extracts have also been proposed for use in immunotherapy. See Juniper, et al. "Polyethelene Glyco - Modified Ragweed Extract: Comparison of Two Treatment Regimens". J. Allergy Clin Immunol 78: 851-6 (1986). See also U.S. Pat. No. 4,180,562 (Patterson) which discloses the use of ragweed polymerized extracts in immunotherapy.
Liposomes have been used in the prior art as inert carriers for drugs, enzymes, hormones and DNA. Liposomes are prepared by dispersing lipids in excess water. The lipids then assemble into spherical lipid particles called liposomes. Each phospholid molecule contains a hydrophilic (water seeking) and a hydrophobic (water avoiding) component. Therefore, when purified phospholipids are mixed with water, they spontaneously organize into bilayer structures.
Liposomes are prepared from phospholipids (e.g., sphingomyelin, lecitin and phosphatidyletanolamine) in combination with cholesterol and charged lipids such as dicetyl phosphate and stearylamine.
In the aqueous regions of a liposome, various salts, small molecules and large molecules can be trapped or encapsulated during liposome synthesis. The encapsulation of small molecules by liposomes allowed their early use as models of natural membrance functions such as complement fixation. Liposomes that are compromised by antibody and complement release the entrapped molecules, such as glucose, enzyme substrates, spin labels and chromogenic or fluorescent dyes, which can be detected by analytical methods.
Target-specific liposomes have been used as inert carriers for drugs, enzymes, hormones, DNA antigen antibodies and other biochemically important substances. Ho, R.J.Y., Rouse, B.T.; Huange, L., "Target Sensitive Liposomes Preparation and Characterization", Biochemistry 25: 5500-5506, 1986; Connor, J., Sullivan, S.M., & Huang, L. "Monoclonal Antibody and Liposome", Pharmacol. Ther. 341-365, 1985. Many techniques have been used for binding of hydrophilic proteins to liposomal surfaces. Torchilin, V.P. (1983): Targeted Drugs (Goldberg, E., ed.) pp. 127-152; Van Rooijen, N. and Van Nieuwmegen, R. (1981): Targeting of Drugs (Gregoriadis, G. ed.) Plenum, New York, NATO ASI, Series A. Among the immobilization techniques covalent modification of proteins with hydrophobic compounds which may serve as an anchor to lipid membranes seems to be the most effective. Torchilin, V.P. and Klibanov, A.L. (1981) "Preliminary `Hyrdrophobization` of Hydrophilic Proteins Increase its Binding with Liposomes", Enz. Microbiol. Technol. 3, 297-304; Loelsch, R., Lasch, J., Klibanov, A.L. and Torchilin, V.P. (1981) Acta Biol. Med. Germ. 40, 331-335. In some cases the stable bilayer conformation can be obtained under physiological conditions by the addition of a second lipid component. Rand, R.P., Tinker, D.O. and Fast, P.G. (1971) Chem. Phys. Lipids 6, 333-342 or by the addition of certain transmembrane proteins such as glycoproteins. Ho, R.J.Y. and Huang, L. "Interaction of Antigen Sensitized Liposomes with Immobilized Antibody; a Homogenous Solid Phase Immunoliposome Assay", J. Immunol. 134, 4035-4040, 1985.
Liposomes have been used as vehicle to promote the immunogenicity of many antigens. Allison, A.C. & Gregoriadis, G. (1974) "Liposome as Immunological Adjuvants". Nature (Lond.) 252, 252. Hepatitis B surface antigen-containing liposomes enhance humoral and cell-mediated immunity to the antigen. Hedlung, G., Jansson, B. & Sjogren, H.O. "Comparison of Immune Responses Induced by Rat RT-1 Antigens Presented as Inserts into Liposomes, as Protein Micelles and as Intact Cells"; Immunology, 53, 69. The mechanisms by which liposomes enhance the immunogenicity of an antigen are likely to vary within different antigenic models; (i) the antigen could be present within the aqueous phase, i.e., encapsulated, and liposomes would represent a closed carrier bag ensuring a specific and efficient delivery to their natural in vivo targets, the macrophages. Alving, C.R. & Richards, R.L. (1983) "Immunologic Aspects of liposomes": The Liposomes (ed. M. Ostro), p. 10 Marcel Dekker, New York, Manesis, E.K., Cameron, C.H. & Gregoriadis, G. (1979) Hepatitis B Surface Antigen-Containing Liposomes Enhance Humoral and Cell-Mediated Immunity to the Antigen; FEBS Lett. 102, 107; Hedlung, G., Jansson, B. & Sjogren, H.O. "Comparison of Immune Responses Induced by Rat RT-1 Antigens Presented as Inserts into Liposomes, as Protein Micelles and as Intact Cells", Immunology. 53, 69. (ii) the liposomes could present the antigen to the immune system on a membrane-like structure the antigen is intimately associated to the lipidic lamellae. Gerlier, D., Bakouche, O., & Dore, J.F. (1983), "Liposome as a Tool to Study the Role of Membrane Presentation in the Immunogenicity of a MuLV-related Tumor Antigen", J. Immunol. 131, 485, or when the protein is covalently linked to the phospholipid bilayers. Snyder, S.L. & Vannier, W.E. (1984), "Immunologic Response to Protein Immobilized on the Surface of Liposomes Via Covalent Azo-bonding, Biochem. Biophys Acta, 772, 288; (iii) liposomes can act as a carrier in the hapten carrier system. Van Houte, A.J., Snippe, H. & Willers, J.M.N. (1979), "Characterization of Immunogenci Properties of Haptenated Liposomal Model Membranes in Mice", Immunology, 37, 505.
The presence of some cholesterol within the phospholipid bilayers increased the immunogenic properties of GCSAa liposomes as well as that of haptened liposomes. Van Houte, A.J., Snippe, H., Schmitz, M.G.J., & Willers, J.M.N. (1981), "Characterization of Immunogenic Properties of Haptenated Liposomal Model Membranes in Mice, V. Effect of Membrane composition on Humoral and Cellular Immunogenicity", Immunology, 44, 561. The addition of a negatively charged phospholipid as a minor component was followed by an increase in the immunogenicity of the antigen-liposome complex. Heath, T.D., Edwards, D.C. & Tyman, B.E. (1976), "The Adjuvant Properties of Liposomes", Biochem. Soc. Trans. 4, 129.